Nickel’s Role in Pancreatic Ductal Adenocarcinoma: Potential Involvement of microRNAs

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancer types with a limited overall survival rate due to the asymptomatic progression of symptoms in metastatic stages of the malignancy and the lack of an early reliable diagnostic biomarker. MicroRNAs (miRs/miRNAs) are small (~18–24 nucleotides), endogenous, non-coding RNAs, which are closely linked to the development of numerous malignancies comprising PDAC. Recent studies have described the role of environmental pollutants such as nickel (Ni) in PDAC, but the mechanisms of Ni-mediated toxicity in cancer are still not completely understood. Specifically, Ni has been found to alter the expression and function of miRs in several malignancies, leading to changes in target gene expression. In this study, we found that levels of Ni were significantly higher in cancerous tissue, thus implicating Ni in pancreatic carcinogenesis. Hence, in vitro studies followed by using both normal and pancreatic tumor cell lines and increasing Ni concentration increased lethality. Comparing LC50 values, Ni-acetate groups demonstrated lower values needed than in NiCl2 groups, suggesting greater Ni-acetate. Panc-10.05 cell line appeared the most sensitive to Ni compounds. Exposure to Ni-acetate resulted in an increased phospho-AKT, and decreased FOXO1 expression in Panc-10.05 cells, while NiCl2 also increased PTEN expression in Panc-10.05 cells. Specifically, following NiCl2 exposure to PDAC cells, the expression levels of miR-221 and miR-155 were significantly upregulated, while the expression levels of miR-126 were significantly decreased. Hence, our study has suggested pilot insights to indicate that the environmental pollutant Ni plays an important role in the progression of PDAC by promoting an association between miRs and Ni exposure during PDAC pathogenesis.


Introduction
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy, with a 5-year overall survival of less than 8% [1,2]. PDAC patients are symptomless even in the most advanced stages [1]. Systemic chemotherapy with gemcitabine and FOLFIRINOX 5-FU (irinotecan, oxaliplatin, and leucovorin), in combination with radiotherapy is the first-line treatment; however, due to high percentages of toxicity, they present limited efficacy in both elderly and PDAC patients with advanced stages [3,4]. line treatment; however, due to high percentages of toxicity, they present limited efficacy in both elderly and PDAC patients with advanced stages [3,4].
Ni is a naturally occurring element, which is found in water, soils, air, and sediments, while the International Agency for Research on Cancer (IARC) has classified its compounds as a "group 1" human carcinogen [13,14]. The extensive use of Ni in industrial settings complicates human health effects, with the most scrutinised effect being carcinogenesis [15]. Moreover, the molecular mechanisms of Ni-induced carcinogenesis comprise hypoxia-inducible factor pathways and the generation of oxidative stress, which further lead to DNA damage and the targeting of DNA repair pathways [15,16]. A previous study has suggested a link between Ni content and PDAC [17], while increasing evidence has suggested Ni involvement in several pancreas-related dysfunctions [18].
The loss of PTEN function has been reported as an underlying cause of PDAC development [23,24]. The loss of PTEN function removes the inhibitor input on AKT, thus increasing AKT activity, leading to tumour development. Numerous investigators have reported a loss of p53 function via a loss or mutation of the protein in PDAC tissue, implicating this loss of function as an underlying cause of cell migration and metastasis [25,26]. Ni compounds have been implicated in an increase in oxidative stress. The overactivity of PARP has been suggested to play an important role in PDAC progression following oxidative stress, which promotes cell necrosis and pro-inflammatory gene expression, contributing to cancer development. PARP inhibitors are being investigated as potential anti-cancer therapies, and evidence suggests that mutated PARP may be a factor in the development of BRCA-mutant PDAC [27,28]. PARP inhibitors may have utility through AKT activation, as well as increased apoptosis and mitochondrial protection during oxidative stress [29]. The β-catenin/FOXO system activity is enhanced in the presence of free radicals [30]. Elevated oxidative stress has been implicated in PDAC development, and the relationship between FOXO1 activity and the Wnt/β-catenin system is critical for maintaining normal cellular function. The cellular loss of FOXO1 results in the presentation of stem-like cells that promote the development of PDAC [31,32]. The FOXO1-mediated inhibition of PDAC formation is partially due to the inhibition of β-catenin activity, which promotes tumour formation [33]. FOXO1 activity is negatively moderated by miRs (miR-21 and miR-27a), which are upregulated in PDAC. Increased miR-21 and miR-27a activity decreases FOXO1 expression/activity, effectively preventing FOXO1's control of β-catenin [34,35]. The regulation of proteins associated with cellular function, growth, and proliferation is complex and subject to multiple regulatory influences. Therefore, in this study, we underline the foundation of the mechanistic effects of Ni on the cellular function of PDAC by examining different proteins involved in either apoptosis, cell proliferation, or both ( Figure 1).
Our previous studies have shown that environmental toxins, such as Cd, can promote their toxicity through miRs [12,18,60]. Toxic metals such as Ni have not been studied as extensively as Cd, and, therefore, only a small number of miRs associated with Ni exposure has been characterized. The functions and mechanisms of these miRs in carcinogenesis remain to be elucidated. Therefore, our aims were to detect Ni concentrations in both human tumour and non-tumour tissue samples, assess NiCl 2 -and Ni-acetate-mediated toxicity during PDAC progression, and investigate the role of miRs in response to NiCl 2 exposure. In the current study, a newly found role of miRs was studied in Ni-induced PDAC carcinogenesis, and the cellular and molecular changes in the control and PDAC cell lines following exposure to NiCl 2 or Ni-acetate were also examined.

Study Population and Sampling
A case-control study was conducted at the First Surgical Clinic, University Clinical Center of Serbia, and the Faculty of Pharmacy, University of Belgrade. Twenty-eight patients with histopathologically confirmed exocrine pancreatic carcinoma were involved in the study. No exclusion criteria regarding the age, sex, or cancer stage were set. All patients were citizens of the Republic of Serbia and were enrolled in the study between May 2014 and December 2016, prior to receiving any chemotherapy or radiotherapy. After the study aim, procedures, and risks were explained to the patients, each enrolled patient confirmed they understood everything and consented to participate in the study. Samples of the cancerous tissue (approximately 1 g each) were obtained by excision during Whipple procedure or total pancreatectomy at the First Surgical Clinic, University Clinical Center of Serbia. Twenty samples were taken as controls during routine post-mortem examination of twenty patients whose death did not occur due to cancer at the Department of Forensic Medicine, Faculty of Medicine, University of Belgrade. A Forensic Medicine Specialist confirmed the absence of malignancies in control samples. Fresh, untreated control samples were frozen and kept at −20 • C. In this way, the test and control and cancer samples were prepared and analyzed identically. The study was approved by the Ethics Committee of the University Clinical Center of Serbia (Approval No. 31/8).

Human Sample Analysis
The exact mass of the samples was determined prior to mineralization. A mix of 7 mL p.a. nitric acid (65% W/V, Merck, Darmstat, Germany) and 1 mL p.a. hydrogen peroxide (30%, W/V, Sigma-Aldrich, Saint Louis, MI, USA) was used for mineralization in a microwave oven (Milestone Ethos One, Milestone, Sorisole, Italy) with SK-10 Teflon rotor and cuvettes rinsed with nitric acid thrice (Milestone, Sorisole, Italy). The samples were heated to 180 • C for 15 min (at 1000 W and 50 bar), then digested for 15 min (at 180 • C, 1000 W, and 50 bar), and cooled to room temperature over 15 min. Mineralized samples were transferred quantitatively to 25-millilitre volumetric flasks and diluted using redistilled water. Ni concentration was determined in the obtained solutions. The blank was prepared by following the same steps, only without a tissue sample. Ni concentration was determined using Agilent 200 Series AA AAS (Agilent Technologies, Santa Clara, CA, USA), using an Agilent AAS GTA120 graphite cuvette (Agilent Technologies, USA) against the blank. A series of standard solutions was prepared by diluting 1000 mg/mL nickel (II) nitrate solution (Merck, Darmstat, Germany) with redistilled water.

LC50 Assays to Determine Nickel Toxicity after 48 h of Exposure
Each of the five cell lines was plated as it has been previously described [60]. Cells were incubated for 48 h with increasing concentrations of the Ni compounds or vehicle (12 concentrations, 0-1 mM). After exposure, an 11-millimolar stock MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) stock solution was prepared in PBS (1.1 mM of final concentration in each well) and added to each well. Plates were returned to the incubator (37 • C/5% CO 2 ) for 4 h. A total of 50 µL of DMSO was added to each well to solubilize the formazan crystals and the plate incubated for an additional 30 min at 37 • C. Viable cells were then measured by the absorbance at 540 nm (BioTek-HT plate reader, Agilent, Santa Clara, CA, USA), which is directly proportional to the number of live cells.

Changes in Protein Expression
The measurement of β-catenin was determined using a commercially available kit for measuring human β-catenin in cell lysates (MyBioSource, Inc. #MBS266009; San Diego, CA, USA) and used as it has described in our previously published data based on the manufacturer's protocol [60]. β-catenin was quantified by measuring the absorbance at 450 nm and extrapolating to the standard curve.

Caspase 3/7 Kinetic Assays
Caspase 3/7 activity (measurement of apoptosis) was measured by using the commercially available kit caspase 3/7 conversion of the non-fluorescent substrate Z-DEVD-R110 bis-(N-CBZL-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide;) to the rhodamine 110 leaving group, which fluoresces at 499 nm ex /521 nm em (Apo-One™ Homogeneous caspase-3/7 assay, Promega, Madison, WI, USA). The fluorescence generated was directly proportional to the amount of caspase 3 and 7 present. Cells were maintained and plated as described above. Exposure was initiated by the addition of 50 µM NiCl 2 , Ni-acetate or vehicle for 48 h in a 37 • C/5% CO 2 incubator. After exposure, caspase 3/7 activity was determined as it has described previously [60]. Emitted fluorescence was measured using a Bio-Tek ® plate reader and Gen5 software at 485 ex /530 em .

Statistical Analysis
IBM SPSS (v18.0, IBM Corporation, Armonk, NY, USA) was used to analyse Ni tissue content. Ni concentration was determined from a standard curve; subsequently, Ni content in fresh tissue samples in ng/g was calculated. Kolmogorov-Smirnov test was used to test data normality, then the data were analysed using Mann-Whitney U-test (p < 0.05 was considered significant). GraphPad Prism version 9.03 (GraphPad Software, San Diego, CA, USA) was used for other statistical analyses and preparation of graphs. t-test was utilized together with Tukey's post hoc analysis to examine miRs expression levels in response to NiCl 2 treatment. Experiments were carried out in triplicates for miRs analysis. miR-related data were analysed using GraphPad Prism (v 9.1-9.3; San Diego, CA). Analyses included: (1) one-way ANOVA with the specialised Dunnett's test for post hoc comparison to control values, (2) two-way (cell type x treatment) or three-way (cell type x treatment x protein) ANOVA with Tukey's test adjusted for multiple comparisons or Sidak's post hoc comparison test. The performed analyses are listed in the results section, or the appropriate figure legend. LC50 values were determined by fitting the data to a nonlinear curve fitting, log concentration x response (three-parameter) single-site model, to yield a sigmoid inhibition curve. All data, except for Ni content assays, are expressed as the mean ± SD of 4-8 assays performed in duplicate or triplicate where indicated. For data that violated the assumption of normal distribution, the nonparametric Kruskal-Wallis test was performed, followed by a post hoc comparison using the corrected Dunn's test. Significance is set at α = 0.05.

Ni Concentration in PDAC Tissue Samples
In total, 28 patients with histological PDAC diagnosis (cases) and 20 death cases unrelated to cancer (controls) showed Ni levels. After the tissue mineralization, graphite furnace atomic absorption spectrometry was used to determine the Ni concentrations. As shown in Figure 2, the Ni levels in the cancerous tissue (1.46-192.10 ng Ni/g wet tissue) were significantly higher than the control levels (1.22-24.06 ng Ni/g). According to the Kolmogorov-Smirnov test, median values were used since the collected data did not follow a normal distribution. Median Ni concentration in control tissue was 8.88 ng/g. Median Ni concentration in cancerous tissue was 31.54 ng/g and was statistically significantly higher than in control tissue (p < 0.01).

Determination of LC50 Values for NiCl2 and Ni-Acetate in PDAC Cell Lines
We determined the LC50 values for NiCl2 and Ni-acetate in multiple PDAC cell lines. The initial LC50 analysis established baseline values for subsequent experimentation. Each cell line was incubated in its optimum growth media and exposure started with the addition of increasing concentrations of NiCl2 ( Figure 3A-C) or Ni-acetate ( Figure 4A-C). The plates were returned to the incubator (37 °C/5% CO2) for 48 h. There was a reduction in cell viability with increasing concentrations of either NiCl2 ( Figure 3A) or Ni-acetate ( Figure 4A). The shapes of the curves were best fitted to a single site model (three parameter model). When examining the curves, the HPNE and AsPC-1 cell lines were shifted to the left (lower LC50), followed by the Panc-1 and BxPC-3 cells, with the Panc-10.05 and MiaPaCa-2 cells exhibiting LC50 values to NiCl2 of >5000 μM and 4209 μM, respectively ( Figure 3A). The rank order for NiCl2 LC50 values was AsPC-1 < HPNE < BxPC-3 = Panc-1 << Panc-10.05 = MiaPaCa-2. These differences in LC50 values are reflected in Figure 3B and Table 1. For NiCl2 exposure, the Kruskal-Wallis analysis revealed a significant difference in LC50 values between cell lines H(6) = 41.64; p < 0.0001. The differences between the groups were determined with Dunn's test for multiple comparisons, as shown in Figure 3B and Table 1. The maximum lethality, at 10 mM NiCl2, demonstrated a significant effect across the cell lines H(6) = 37.72; p < 0.0001 ( Figure 3C). Interestingly, BxPC-3 appeared to be the most sensitive cell line, with <10% viability at 10 mM NiCl2. The Panc-10.05 and MiaPaCa-2 cells appeared to have the greatest resistance to NiCl2 toxicity. with nearly 25-30% cell viability at 10 mM NiCl2. The HPNE control cells exhibited only modest (19%) resistance to the toxicity of NiCl2.

Determination of LC50 Values for NiCl 2 and Ni-Acetate in PDAC Cell Lines
We determined the LC50 values for NiCl 2 and Ni-acetate in multiple PDAC cell lines. The initial LC50 analysis established baseline values for subsequent experimentation. Each cell line was incubated in its optimum growth media and exposure started with the addition of increasing concentrations of NiCl 2 ( Figure 3A-C) or Ni-acetate ( Figure 4A-C). The plates were returned to the incubator (37 • C/5% CO 2 ) for 48 h. There was a reduction in cell viability with increasing concentrations of either NiCl 2 ( Figure 3A) or Ni-acetate ( Figure 4A). The shapes of the curves were best fitted to a single site model (three parameter model). When examining the curves, the HPNE and AsPC-1 cell lines were shifted to the left (lower LC50), followed by the Panc-1 and BxPC-3 cells, with the Panc-10.05 and MiaPaCa-2 cells exhibiting LC50 values to NiCl 2 of >5000 µM and 4209 µM, respectively ( Figure 3A). The rank order for NiCl 2 LC50 values was AsPC-1 < HPNE < BxPC-3 = Panc-1 << Panc-10.05 = MiaPaCa-2. These differences in LC50 values are reflected in Figure 3B and Table 1. For NiCl 2 exposure, the Kruskal-Wallis analysis revealed a significant difference in LC50 values between cell lines H(6) = 41.64; p < 0.0001. The differences between the groups were determined with Dunn's test for multiple comparisons, as shown in Figure 3B and Table 1. The maximum lethality, at 10 mM NiCl 2 , demonstrated a significant effect across the cell lines H(6) = 37.72; p < 0.0001 ( Figure 3C). Interestingly, BxPC-3 appeared to be the most sensitive cell line, with <10% viability at 10 mM NiCl 2 . The Panc-10.05 and MiaPaCa-2 cells appeared to have the greatest resistance to NiCl 2 toxicity. with nearly 25-30% cell viability at 10 mM NiCl 2 . The HPNE control cells exhibited only modest (19%) resistance to the toxicity of NiCl 2 .
There was a reduction in cell viability with increasing concentrations of Ni-acetate ( Figure 4A). The shapes of the curves were best fitted to a single site model (three parameter model). The chemical form of Ni appeared to have a robust effect with the LC50 for all the cell lines, except Panc-1, which was significantly reduced when exposed to Ni-acetate compared to the LC50 values for NiCl 2 . The largest reduction was observed in the anc-10.05 cells, which changed from >5000 µM to 183.5 µM, followed by the MiaPaCa-2 cells, which had an LC50 value for NiCl 2 in excess of 4200 µM to 119 µM for Ni-acetate. The potency series following Ni-acetate exposure was AsPC-1 < MiaPaCa-2 ≤ HPNE ≤ Panc-10.05 < BxPC-3 << Panc-1. These differences in LC50 values are reflected in Figure 4B and lines H(6) = 32.66; p < 0.0001. The differences between the groups were determined with Dunn's test for multiple comparisons and are shown in both Figure 4B and Table 1. The maximum lethality at 10 mM Ni-acetate was determined and we observed a significant effect across the cell lines H(6= 37.27; p < 0.0001 ( Figure 4C). Similar to our observation following NiCl 2 exposure, the BxPC-3 cell line was the most sensitive to the toxic effects of Ni-acetate, with < 10% viability at 10 mM N-acetate. The Panc-10.05, MiaPaCa-2, and AsPC-1 cells appeared to have the greatest resistance to Ni-acetate toxicity, with approximately 25-30% cell viability at 10 mM Ni-acetate. The HPNE control cells exhibited only modest (19%) resistance to Ni-acetate toxicity, which was similar to the outcome after exposure to NiCl 2 .        We then examined the mechanistic effects of Ni on the cellular function of PDAC by assessing key proteins. Briefly, the cells were exposed to 50 µM NiCl 2 or Ni-acetate for 48 h and the quantity of β-catenin was measured using a whole-human-cell assay kit ( Figure 5A). Exposure to the Ni compounds did not result in robust changes in β-catenin content in any of the cell lines, although there was a slight downward trend in the Panc-10.05 cell line. The quantity of β-catenin was significantly influenced by the cell line (F 4,30 = 20.37; p < 0.0001). The basal content (control) of β-catenin was approximately 40% higher in the Panc-1 cells compared to the HPNE control cells (p < 0.01). ELISAs were used to determine the protein content in the PDAC cell lines. The measurement of relative phospho-AKT is presented in Figure 5B. There were significant differences between the cell lines (

Changes in Protein Expressions of PTEN and PARP and Caspase 3/7 Activity
The cells were incubated with 50 µM NiCl 2 or Ni-acetate for 48 h, and the expression of PTEN ( Figure 6A), PARP ( Figure 6B) was quantified. The PTEN expression was dependent on the cell line (F 4,45 = 65.3; p < 0.0001), with higher levels of basal expression in the HPNE control group compared to each of the PDAC cell-line controls (p < 0.001). Exposure to 50 µM NiCl 2 significantly increased the PTEN expression in the Panc10.05 cells compared to their vehicle control (p < 0.01). The expression of PARP exhibited an inverse profile compared to what was observed with the PTEN expression (F 4,45 = 5.33; p = 0.004). The HPNE control cells had significantly lower PARP expression than each PDAC cell-line control group (p < 0.001). The treatment significantly impacted the activity of caspase ( Figure 6C), as did the cell line (F 4,105 = 2.79; p = 0.0299) with a trend towards reductions in caspase activity (F 2,105 = 9.65; p = 0.0001). Exposure to NiCl 2 resulted in small, nonsignificant, reductions in caspase 3/7 activity. Exposure to Ni-acetate resulted in larger reductions in caspase 3/7 activity, with a significant (p < 0.01) 20.6% reduction in the HPNE group. The expression of cleaved (active) caspase 3 ( Figure 6D) was significantly (F 4,45 = 27.79; p < 0.0001) dependent on the cell line, with a minimal effect of treatment. Only the Panc-1 control group exhibited significantly (p < 0.01) lower expression than the HPNE control group.

Changes in Protein Expressions of PTEN and PARP, and Caspase 3/7 Activity
The cells were incubated with 50 μM NiCl2 or Ni-acetate for 48 h, and the expression of PTEN ( Figure 6A), PARP ( Figure 6B  caspase activity (F2,105 = 9.65; p = 0.0001). Exposure to NiCl2 resulted in small, non-significant, reductions in caspase 3/7 activity. Exposure to Ni-acetate resulted in larger reductions in caspase 3/7 activity, with a significant (p < 0.01) 20.6% reduction in the HPNE group. The expression of cleaved (active) caspase 3 ( Figure 6D) was significantly (F4,45 = 27.79; p < 0.0001) dependent on the cell line, with a minimal effect of treatment. Only the Panc-1 control group exhibited significantly (p < 0.01) lower expression than the HPNE control group. activity was significantly (p < 0.01) reduced after exposure to Ni-acetate. Data are expressed as the mean ±SD of four (protein expression) or eight (caspase 3/7 activity) assays performed in duplicate (** p < 0.01; *** p < 0.001).

Changes in miR Expression Levels in Response to Nickel Chloride Treatments in PDAC Cells
After investigating the mode of action of NiCl2 in apoptosis, we further analysed its effect on miR expression by using the Panc-1 and MiaPaCa-2 PDAC cell lines as a model, based on our previous studies [41,60]. The miR-221, miR-155, and miR-126 are closely linked to PDAC progression in different stages [41,54,62,63]. Following incubation, NiCl2, miR-221 expression was elevated both in Panc-1 and MiaPaCa-2 cells (385-fold, p < 0.001, and 5.8-fold, p < 0.05, respectively; Figure

Changes in miR Expression Levels in Response to Nickel Chloride Treatments in PDAC Cells
After investigating the mode of action of NiCl 2 in apoptosis, we further analysed its effect on miR expression by using the Panc-1 and MiaPaCa-2 PDAC cell lines as a model, based on our previous studies [41,60]. The miR-221, miR-155, and miR-126 are closely linked to PDAC progression in different stages [41,54,62,63]. Following incubation, NiCl 2 , miR-221 expression was elevated both in Panc-1 and MiaPaCa-2 cells (385-fold, p < 0.001, and 5.8-fold, p < 0.05, respectively; Figure 7A,D; n = 3 for both). Similarly, an upregulation of miR-155 was noted in both the Panc-1 (28.5-fold) and MiaPaCa-2 (151-fold) cells after NiCl 2 exposure ( Figure 7B,E; n = 3, p < 0.05 for both), while NiCl 2 treatment reduced miR-126 expression considerably in Panc-1 (96.6-fold) and MiaPaCa-2 (100-fold) ( Figure 7C,F; n = 3, p < 0.0001 for both). Our results suggest that NiCl 2 , increases oncogenic miR expressions, such as miR-221 and miR-155, while reducing the expression levels of the tumour suppressor, miR-126. To date, this is the first analysis to show that NiCl 2 could influence the expression levels of these miRs in PDAC. n = 3, p < 0.0001 for both). Our results suggest that NiCl2, increases oncogenic miR expressions, such as miR-221 and miR-155, while reducing the expression levels of the tumour suppressor, miR-126. To date, this is the first analysis to show that NiCl2 could influence the expression levels of these miRs in PDAC.

Discussion
The primary results of this study are that (1) a positive correlation between Ni concentration and the incidence of PDAC was found in the PDAC tissue samples; (2) exposure to Ni was toxic to all the PDAC cells and, although Ni-acetate appeared to be most toxic, both NiCl 2 and Ni-acetate alter the expression of phospho-AKT, PTEN, and FOXO-1; (3) the oncogenic miR-221, miR-155, and tumour suppressor miR-126 were highly dysregulated in the PDAC cells treated with NiCl 2 compared with the control PDAC cells.
Ni can stimulate global hypermethylation, which results in the suppression of key tumour-suppressor genes, the activation of proto-oncogenes, and senescence [64]. Oxidative stress is another important route for Ni-induced carcinogenesis [65], while the hypoxiainducible signaling pathway is a key mechanical route for Ni-induced carcinogenesis, as the transcription factor hypoxia-inducible-factor-1 (HIF-1) is triggered [66].
Our current study provides some pilot insights into Ni concentration and incidence of PDAC based on in vivo data. Ni concentrations determined in this study are comparable to those observed in other studies. Namely, Ni concentration determined in organ samples obtained post-mortem from subjects without pancreatic cancer were ranging from 1 to 19 ng/g [67]. Higher Ni content in tissue samples obtained from cancer patients has been observed in other studies. Depending on the biological material sampled, conflicting results have been published, mainly relying on clipped toenails as biological material [68]. However, recent reviews have suggested that toenails were likely inadequate biological material for trace element assessment [69]. A similar concern regarding using toenails was made years ago by Vinceti et al. (2007) when assessing the relationship between Cd content and prostate cancer in humans [70]. Furthermore, several recent studies tend to consider agent content at cancer sites rather than in other tissues [71][72][73]. Several studies have identified that Ni content in PDAC tissue is a reliable biomarker across different studies [67,74,75], while our results also suggest that Ni could be involved in PDAC development in humans.
Studies examining the impact of exposure to various environmental factors on the development of pancreatic cancer are often limited by the relatively small number of cases of pancreatic cancer and the ability to evaluate a small number of environmental factors in studies [76][77][78]. However, a large clinical case study and follow-up indicated the possibility of an association between PDAC risk and Ni exposure [79]. The incidence of pancreatic cancer has been associated with occupational exposure to Ni in several studies [80][81][82][83] and one meta-analysis [84].
The disruption of bio-element homeostasis may be another potential mechanism of Ni carcinogenicity. Iron (Fe)-cell homeostasis disorders were found in our set of PDAC patients when compared to control tissues through a positive correlation between Ni and Fe pancreatic levels (unpublished data). Ni ions have been found to inhibit the histone class, H3K9 demethylase, whose activity depends on Fe and 2-oxoglutarate and increases H3K9 dimethylation [85]. Ni ions compete with Fe ions, which are physiologically linked to the JmjC domain of dioxygenases, enzymes that have catalytic activity [86]. The same study estimated that since Ni ions have a higher affinity with this site, once Ni ions replace Fe ions in this domain, the Fe ions cannot bind again. Oxidative DNA, lipid, and protein damage are significantly increased in the presence of Fe [87,88]. Considering the above results, elevated Ni levels in the body may cause a disturbance of Fe homeostasis, thereby increasing the concentration of this bio-element; this can be a starting point for a further cascade of events that leads to the development of pancreatic cancer. Another recent study suggested that the role of Ni should be further investigated by considering its possible epigenetic effects [18]. The study implied that Ni could be involved in the malignant transformation of pancreatic cell cultures, but also highlighted the importance of toxicants affecting the cells at the same time as Ni, as the endpoint seemed to depend on the presence of other toxicants, as well as Ni concentration. Hence, the precise molecular mechanisms of the role of Ni in PDAC should be addressed through in vitro investigations.
The goal of this paper was to examine not only the transformation of normal cells into cancerous cells, but also the potential for Ni compounds to promote tumour growth in PDAC cell lines. We chose to investigate the effects of Ni exposure at the cellular/molecular level, with higher concentrations of the toxic metal, or longer durations of exposure. It has been reported that Ni at higher concentrations can halt the cell cycle and stop proliferation. In vivo studies have used extremely high doses, up to 900 mg/kg [89], but these doses far exceed what would be considered the upper limit of human exposure, which is 1 mg/day. Evidence from the in vivo studies suggests that Ni stops cell growth in the G 2 /M phase and induces apoptosis through a p53-regulated pathway [89,90]. Other data suggest that Ni-mediated anti-tumour characteristics are mediated by a reduction in angiogenesis, reducing the vasculisation of tumours [91]. A growing body of evidence suggests that Ni and Ni-containing compounds are carcinogenic, a fact recognized by international organisations [13]. The toxicity and carcinogenicity of Ni is supported by our previous data [10,11,18,60], as well as other reports using different cell-line models representing different cancers [92][93][94][95]. The similarity between toxic mechanisms and our work in PDAC cell lines suggests that Ni may work through the Akt pathway, which is independent of FOXO1 activation [92,94]. Using a small-cell carcinoma cell model, it was evident that Ni-induces the activation of VEGF, increasing angiogenesis and the vasculation of the tumour. The activation of VEGF involved Akt, but not FOXO1 [93]. Ni toxicity appears to partially depend on the cell line. Comparing mouse embryo fibroblasts (BALB/3T3) and hepatic tumour cells (HepG2), HepG2 cells appear to have greater sensitivity to the effects of nickel chloride up to 1 mM [95].
Our long-term goal is to study the entire continuum of Ni-induced toxicity. HPNE cells represent 'normal' or 'control' cells, and we were able to determine the toxicity of NiCl 2 and Ni-acetate as well as cellular/molecular changes following exposure to Ni. The tumour cell lines vary according to the mutations that are characterized. For example, all cell lines express a TP53 mutation. Only AsPC-1 cells express the FBXW7 mutation. Panc-10.05 exhibits the fewest mutations (TP53 and KRAS), but this cell line appears to be the most sensitive to the effects of Ni. Panc-1 and BxPC-3 cells express the same mutations in TP53, CDKN2A, MAP2K4 and SMAD4. MiaPaCa-2 cells exhibit a deletion of CDKN2A and a mutation in KRAS, and TP53. One question we addressed was whether these deletions have a role in Ni-mediated toxicity. Not only could Ni result in toxic effects and transform a normal, control cell into a cancerous cell, but it may also accelerate tumour growth in tumour cells. By performing LC50 analysis, we were able to visualise any growth/viability changes, even at low (<1 µM) concentrations. We did not observe any effect that would suggest Ni-mediated increase in cell growth.
Lethality curves revealed LC50 values for NiCl 2 ranging from 196 µM to over 5000 µM, depending on the cell line. Ni-acetate increased (leftward shift of the curve) the lethality in most cell lines, resulting in an LC50 range of 83-1,126 µM, suggesting the increased lethality of the organic Ni form. The values we report are similar in magnitude to those reported in β-cells exposed to NiSO 4 [96]. We did consider that acetate may change the pH of the media by conversion to acetic acid. The media did contain phenol, and there were no significant changes in the pH of the media, suggesting that pH was not the cause. Furthermore, it may be possible that acetate itself, being a cellular metabolite, has an effect. We have examined the literature and found nothing that would support this hypothesis. In studies examining carcinoma cell lines, the use of acetate can be toxic or promote apoptosis, but only at very high concentrations. In oligodendroglioma cell lines, investigators used 12 mM and 36 mM concentrations of sodium acetate and observed 93+% viability at 12 mM. Our maximum concentration was 10 mM of nickel acetate, suggesting that a reduction in viability was not due to the acetate [97]. Another study found that concentrations of sodium acetate that are comparable to the cellular concentrations generated by complete glyceryl triacetate catalysis promoted growth arrest over an exposure time of 24 h. In HCT-15 and RKO cell lines (colorectal carcinoma), 10 mM acetate reduced viability by less than 10%. Other studies by the same investigators used 70 and 140 mM sodium acetate before significant reductions in viability were observed. An increase in apoptosis, but not necrosis, was reported after 48 h exposure to sodium acetate [98]. In cultured human gastric adenocarcinoma epithelial cells, incubation with up to 12.5 mM sodium acetate for 72 h increased cell viability and proliferation, contrary to what would be expected. The increase in viability and proliferation was concentration-dependent. Concentrations of sodium acetate greater than 12.5 mM inhibited cell growth in a dose-dependent manner by up to 50%. [99].
The most prevalent difference in protein expression/activity was between cell lines. NiCl 2 increased the expression of phospho-Akt 146% in the BxPC-3 and 83% in the Panc-10.05 cell lines. Ni-acetate increased phospho-Akt expression by 77% in the Panc-10.05 cell line. Both NiCl 2 and Ni-acetate reduced the expression of FOXO1 in the Panc-10.05 cells by 51% and 19%, respectively. The PTEN expression was elevated by 15% in the Panc-10.05 cells after NiCl 2 exposure. Exposure to NiCl 2 or Ni-acetate resulted in caspase 3/7 activity trending downward, with only the HPNE cells demonstrating a significant reduction (43%) in the Ni-acetate group. Collectively, there were minimal effects of either NiCl 2 or Ni-acetate after 48 h exposure on protein expression or activity. It appears that the Panc-10.05 cell line was most affected by the Ni compounds and in particular, the expression of phospho-Akt and PTEN.
Chickens fed 300-900 mg/kg NiCl 2 for 14 days demonstrated a generalised increase in hepatic apoptosis with elevated expression of caspase 3, 8, and 9, PARP, and p53, all of which were time-and dose-dependent [90]. The lower dose effects were small after 14 days, but the conclusion was that NiCl 2 enhanced apoptosis via mitochondrial, Fas, and endoplasmic-reticulum-dependent pathways. Similar results were observed in chicken kidneys receiving the same dosing regimen as the hepatic studies [100]. Similar activation of caspase 3, 7, and 9, as well as increased PARP degradation, was reported in β-cells [96]. The studies by Wu et al. (2011) focused solely on protein content and not activity. In a mouse model, the administration of 7.5, 15, or 30 mg/kg NiCl 2 resulted in a 70% reduction in the ratio of p-AKT/AKT and a reduction of nearly 40% in mRNA expression in the kidney. The authors concluded that NiCl 2 -induced autophagy took place via the PI3K/AKT/mTOR pathway by reducing the production and activation of AKT (p-AKT), resulting in mTOR inhibition [96]. Our data demonstrated a significant elevation in p-AKT in HPNE control cells, suggesting an increased activation downstream, potentially activating mTOR. Two of the four tumour lines exhibited effects associated with Ni exposure, with both the BxPC-3 and Panc-10.05 cells showing increased p-AKT content after exposure to NiCl 2 , but with no effect of Ni-acetate. A transformed cell line was used to examine the upstream control of AKT via PTEN. Ni was included in the media for two months to transform the cell line, before different cell growth and apoptotic markers were examined. The transformed cells exhibited a significantly higher expression of p-AKT, with reductions in GSK3β and free radical formation [94]. The elevated levels of p-AKT were not due to changes in PTEN regulation (no change in content), but likely due to the PI3K activation of AKT.
Interestingly, in one study, although it was expected that GSK3β would be upregulated and that Wnt/β-catenin would be downregulated, there was no change in β-catenin content or activity. The authors also state that no changes in p53 were observed [101]. These slight differences compared with our study could be due to the different cell lines utilised, and most of the changes observed by the authors were with exposure times greater than 48 h, which was our duration of exposure. Shorter exposures using NiSO 4 in HepG2 cells resulted in a biphasic AKT response, with an initial increased expression, followed by reduced expression with increasing time [92]. The biphasic effects of Ni on the phosphorylation of AKT were also observed in a different cell model; these effects peaked after about 15 h and began to decline after 24 h [93]. Eckers et al. (2009) also reported downstream changes in the levels of phosphorylation of FOXO1, but not in FOXO1 expression, after 5 min of exposure, up to 60 min [92]. In vivo studies administered Ni over the course of 2 weeks, and up to 6 weeks [89]. The longer cell exposure times to Ni could underscore some of the differences observed in protein expression. The involvement of the AKT/FOXO system is necessary for normal apoptotic function at the cellular level. In our current study, only the Panc-10.05 cells demonstrated a significant reduction in FOXO1 expression. Reduced FOXO1 would remove the inhibition exerted on the Wnt/β-catenin system.
Recent studies have mentioned that specific miR expression profiles could be not only used as promising non-invasive diagnostic biomarkers [101], but also that several miRs play a significant role in metal carcinogen-induced cell malignant transformation and tumorigenesis [102]. Nickel can interfere with miR networks to degrade mRNA or block protein synthesis [103]. Specifically, a previous study indicated that NiCl 2 exposure could induce the upregulation of miR-210 with the stabilization of the HIF-1α protein, which leads to alterations in metabolism [104,105]. Furthermore, in a recent review, it was mentioned that Ni exposure can cause aberrations in the expression levels of several miRs, which are thought to play a crucial role in tumour growth, cell transformation, and angiogenesis [106]. Several studies have also suggested that Ni exposure can induce the expression levels of miR-152, miR-203, miR-4417, miR-222, and miR-210 [107]. In our current study, we found that the expression of miRs were considerably dysregulated in PDAC cell lines treated with NiCl 2 compared to corresponding control cells. The miR-221 and miR-155 expression levels were significantly upregulated in the Panc-1 and MiaPaca-2 PDAC cell lines treated with NiCl 2 , while miR-126 was considerably downregulated.
Upregulation of miR-221 expression levels has previously been related with migration, metastasis, and uncontrolled proliferation of PDAC cells [63]. In addition, overexpression of miR-221 can have as an outcome the loss of expression of cyclin-dependent kinases (CDKNs), which is linked to liver fibrosis and unfavourable prognosis of PDAC [107]. It has been recently suggested that the oncogenic effect of miR-221 in PDAC is due to the decrease of PTEN, PUMA, p27 (kip1) and p57 (kip2), which can promote proliferation, inhibit apoptosis and increase the invasive manner of cancer cells [108]. Importantly, when examining the relative expression levels of the selected miRs in our recent study, miR-221 was significantly upregulated in PDAC cells that have been treated with NiCl2 in comparison to the non-treated/control PDAC cells. Furthermore, previous reports have also shown the overexpression of miR-155 during PDAC pathogenesis [52], which is linked to poor prognosis [108]. In our current study, we showed that the expression levels of miR-155 were significantly increased, when PDAC cells were exposed to NiCl2 compared with the control Panc-1 and MiaPaCa-2 cells. A recent study has also mentioned that numerous exosomal miRs such as miR-155, could be potential biomarkers for the early diagnosis of this malignancy [109], while further studies have shown that miR-155 is linked to clinical stage and lymph node metastasis in PDAC patients [110][111][112].
The findings of our current study indicated that the expression levels of miR-126 were significantly downregulated in both Panc-1 and MiaPaCa-1 cells following NiCl2 exposure related to the expression levels of control PDAC cells. Therefore, our recent study promotes some pioneer insights into the correlation between NiCl2 exposure and expression levels of oncogenic and tumour suppressor miRs during PDAC pathogenesis.
In summary, our findings suggest novel roles of NiCl2 on PDAC and specifically highlight that Ni plays a crucial role during PDAC development and underscores the role of miRs in response to NiCl2 during PDAC pathogenesis. Our results have also helped indicate the LC50 values for NiCl2 and Ni-acetate in PDAC cell lines and have provided some of the first information regarding the changes in protein expression in PDAC cells after 48 h exposure.

Conclusions
Prompted by the interesting results obtained in human pancreatic tissue, this is the first study to examine NiCl 2 treatment in PDAC cells. Using 48 h exposure times and PDAC cell lines, we established LC50 values for both NiCl 2 and Ni-acetate. These LC50 values will be used in future research, in which we will use subtoxic concentrations for extended periods, while examining cellular and molecular changes. Most of the observed differences in protein expression were cell-line-dependent, but there were instances, most notably in the BxPC-3 and Panc-10.05 cells lines, where NiCl 2 and Ni-acetate exposure resulted in expression changes. One shortcoming of the present study is that, while investigating the immediate toxic effects following Ni exposure, the 48-h time course of exposure may be too short. The 48-h time course was based on previous studies with other toxic metals, and may have been too short to determine changes in protein expression, which would take longer to manifest. Additional studies would be needed for a longer duration of Ni exposure to determine more robust changes in protein expression. There are multiple miRs involved in regulating cellular function, and miR-21 and miR-27a are reported to regulate the activity of AKT [19][20][21][22]. In this study, Panc-1 and MiaPaCa-2 were treated with NiCl 2 , and the results suggested that the expression levels of the selected miRs were significantly disrupted, including the increasing pro-oncogenic properties of miR-221 and miR-155 and the decreasing anti-oncogenic capabilities of miR-126. Our results report novel regulatory roles of NiCl 2 in PDAC, and especially underline the role of NiCl 2 exposure in the alteration of miR expression levels during PDAC pathogenesis. With emerging attention to miRs and the development of new approaches for understanding miRs, several new mechanisms that lead to PDAC initiation and progression, such as NiCl 2 exposure, were noted. Conclusively, our study provides pioneering insights into the pathogenesis and mechanisms of PDAC, promoting effective prognostic approaches.